1. Field Of The Invention
The present invention relates to making and using apatite crystals, both in vitro and in vivo, and to the mitigation of the effects of hard tissue diseases.
2. Natural Sources Of Apatite
Calcium hydroxyapatites, a complex calcium phosphate (Ca.sub.5 (PO.sub.4).sub.3 OH) in crystalline form, occur naturally as geological deposits and in normal biological tissues, principally bone, cartilage, enamel, dentin, and cementum of vertebrates and in many sites of pathological calcifications such as blood vessels and skin. Essentially none of the geological and biological apatites are "pure" hydroxyapatite since they contain a variety of other ions and cations and may have different ratios of calcium to phosphorous than the pure synthetic apatites. In general, the crystals of pure synthetic apatites, geological apatites and many impure synthetically produced apatites are larger and more crystalline than the biological crystals of bone, dentin, cementum and cartilage.
The terms "crystal" and "crystallite" may be used interchangeably in most respects; "crystallite" simply means a small crystal. A crystal is a homogeneous, solid body of a chemical element, compound, or isomorphous mixture, having a regular repeating atomic arrangement of atoms that may or may not be outwardly expressed by planar faces.
The calcium-phosphate crystals of the bones of essentially all vertebrates have the basic crystal structure of hydroxyapatite as determined by X-ray diffraction. Indeed, the calcium-phosphate crystals of essentially all of the normally mineralized tissues of vertebrates, including enamel, dentin, cementum, and calcified cartilage, have the same general crystal structure. For the purposes of the present invention, these tissues are called "hard tissues".
However, the crystals of calcium phosphate found in hard tissues such as bone also contain other atoms and ions such as acid phosphate groups (H.sub.2 PO.sub.4), and carbonate ions, which do not occur in pure, synthetic hydroxyapatite. There is also good evidence that bone crystals either do not contain hydroxyl groups, or contain only very few such groups (Rey et al. (1995) Hydroxyl groups in bone mineral, Bone 16: 583-586) and is therefore more appropriately referred to as "apatite" rather than "hydroxyapatite." Moreover, many of the carbonate and phosphate groups in bone crystals are, from the structural and physical chemical points of view, unstable and very reactive, thus providing certain physical chemical and biological functional and chemical features important in the formation and dissolution of the crystals in biological tissues.
Recent .sup.31 P-nuclear magnetic resonance spectroscopy studies have demonstrated that the short-range order or environment of the H.sub.2 PO.sub.4 groups in bone crystals are distinctly different than the H.sub.2 PO.sub.4 groups in synthetic apatites and other related calcium-phosphate crystals (Wu, Ph.D. thesis M.I.T., "Solid state NMR study of bone mineral," August 1992). These differences in chemical, structural, and short range order of the bone crystals compared with pure, synthetic hydroxyapatite also reflect significant differences in their reactivity and hence in their potential function in a biological environment.
The crystals of bone, dentin and cementum are very small, irregularly shaped, very thin plates whose rough average dimensions are approximately 10 to 50 angstroms in thickness, 30 to 150 angstroms in width, and 200 to 600 angstroms in length. This results in their having a very large surface area to present to the extracellular fluids which is critically important for the rapid exchange of ions with the extracellular fluids. This "ion-reservoir" function of the inorganic crystals is very important for a number of critical biological functions.
For a description of the determination of the size of bone crystals, see Ziv, V., and Weiner, S. (1994) Bone Crystal Sizes: A comparison of transmission electron microscopic and X-ray diffraction line width broadening techniques. Connective Tissue Research, 30: 165-175; also Azaroff, L. V. (1968) Elements of X-Ray Crystallography, McGraw-Hill; also Hurlbut, C. S., & Klein, C. (1977) Manual of Minerology, 19th ed., John Wiley & Sons. Most investigators of bone structure prefer to rely on measurements of X-ray diffraction reflection line widths. This parameter is directly related to coherence length, that is the average distance between lattice dislocations in a given direction.
Synthetic sources of apatite
Synthetic apatites are highly diverse. For example, the characterization of four commercial apatites was reported by Pinholt, et al., J. Oral Maxillofac. Surg. 50(8), 859-867 (August 1992); J. Cariofac. Surg. 1(3), 154-160 (July 1990) reports on a protein, biodegradable material; Pinholt, et al., Scand. J. Dent. Res. 99(2), 154-161 (April 1991) reports on the use of a bovine bone material called BiO-OSS..TM..; Friedman, et al., Arch. Otolaryngol. Head Neck Surg. 117(4), 386-389 (April 1991) and Costantino, et al., Arch. Otolaryngol. Head Neck Surg. 117(4), 379-384 (April 1991) report on a hydroxyapatite cement; Roesgen, Unfallchirurgie 16(5), 258-265 (October 1990), reports on the use of calcium phosphate ceramics in combination with atogeneic bone; Ono, et al., Biomaterials 11(4), 265-271 (May 1990) reports on the use of apatite-wollastonite containing glass ceramic granules, hydroxyapatite granules, and alumina granules; Passuti, et al., Clin. Orthop. 248, 169-176 (November 1989) reports on macroporous calcium phosphate ceramic performance; Harada, Shikwa-Gakuho 89(2), 263-297 (1989) reports on the use of a mixture of hydroxyapatite particles and tricalcium phosphate powder for bone implantation; Ohgushi, et al., Acta Orthop. Scand. 60(3), 334-339 (1989) reports on the use of porous calcium phosphate ceramics alone and in combination with bone marrow cells; Pochon, et al., Z-Kinderchir. 41(3), 171-173 (1986) reports on the use of beta-tricalcium phosphate for implantation; and Glowacki, et al., Clin. Plast. Surg. 12(2), 233-241 (1985), reports on the use of demineralized bone implants.
apatite-forming-systems
Synthetic calcium hydroxyapatite is formed in the laboratory either as pure Ca.sub.5 (PO.sub.4).sub.3 (OH) or hydroxyapatite that is impure, containing other ions such as carbonate, fluoride, chloride for example, or crystals deficient in calcium or crystals in which calcium is partly or completely replaced by other ions such as barium, strontium and lead. Systems for forming apatite by precipitating solutes are known from the literature.
In these processes, hydroxyapatite precipitates in very finely crystalline form in nearly all solutions.
Some examples of methods for the precipitation of hydroxyapatite, the properties of the apatite product, and possible applications are discussed below. For the purposes of this invention, the term "apatite" signifies any of the forms of apatite in which the hydroxyl groups are replaced by other anions.
It is known from U.S. Pat. No. 4,274,879, for example, to prepare hydroxyapatite by mixing milk of lime with at least 60% phosphoric acid in stoichiometric amounts at temperatures of 80.degree. C.-85.degree. C., and a pH of the reaction solution of about 9.0-11.0 in a continuous reaction. The products obtained are suitable for preparing bone replacement parts by sintering at temperatures of 700.degree. C. They are unsuitable as tooth-cleaning substances on account of their fineness. Additional examples of such methods, products, and applications are disclosed in U.S. Pat. Nos. 4,324,772 and 4,849,193.
Apatites in which the OH..sup.- is replaced with simple anions, including F.sup.-, Br.sup.-, I.sup.-, or carbonate, may be prepared by modifying the process for preparing hydroxyapatite. Apatite derivatives in which calcium is replaced by metal ions, such as paramagnetic, radiopaque, or radioactive metal ions, may also be prepared and used within the scope of the present invention. Useful apatites may also be prepared by replacing phosphate with oxyanions or tetrahedral anions containing radiopaque or radioactive metal species. Stoichiometric pure hydroxyapatite has a Ca:P ratio of 1.67:1. The major impurity found in hydroxyapatite is tricalcium phosphate, Ca.sub.3 (PO.sub.4).sub.2, known as "TCP." This impurity can be detected by deviation from the 1.67:1 Ca:P ratio (for large amounts of impurity) or by X-ray diffraction for impurity levels down to 1 percent. Stoichiometric hydroxyapatite is prepared by adding an ammonium phosphate solution to a solution of calcium/ammonium hydroxide. To minimize the amount of TCP formed, it is important to have excess calcium throughout the addition process.
U.S. Pat. No. 5,460,803 to Ming S. Tung issued Oct. 24, 1995 discloses two apatite-forming-systems. The first system particularly involves the use of amorphous calcium compounds such as: amorphous calcium phosphate, amorphous calcium phosphate fluoride, amorphous calcium carbonate phosphate, and amorphous calcium carbonate phosphate fluoride. The compounds have the highest solubilities, fastest formation rates and fastest conversion rates to apatite among all the calcium phosphates under physiological conditions. Moreover, in the presence of fluoride the amorphous compounds convert rapidly to fluoride containing apatite. In a unique aspect, the method takes advantage of the solvent qualities of ethanol.
In a second apatite-forming-system disclosed by U.S. Pat. No. 5,460,803, the amorphous calcium compounds are formed, in situ, as an intermediate prior to the precipitation of the apatite. The method uses carbonated solutions containing calcium ions, fluoride ions, carbonate ions and phosphate ions, maintained under a pressurized carbon dioxide atmosphere. Under the pressurized carbon dioxide atmosphere, the solutions have a lower pH and are stable. When applied under atmospheric pressure, carbon dioxide escapes, causing the pH to increase. This increase in pH results in a supersaturated solution and ultimately rapid precipitation of apatite.
Effect of electrical charge and stress on in vivo apatite-forming-systems
Hydroxyapatite is piezoelectric; that is, it generates an electric charge when mechanically stressed. The electric signals generated by hydroxyapatite when bone is placed under stress ("bone talk") are detected by nearby bone cells, stimulating them to increase the production of hydroxyapatite. This increase in production of hydroxyapatite due to "bone talk" appears to be part of a feedback mechanism causing bone to be strengthened automatically at points of stress concentration. This feedback mechanism is weakened or interrupted in areas surrounding a bone fracture since stress concentrations at the fracture are typically nil. A similar feedback mechanism seems to control the mineral content of intact bone. When normal "bone talk" is no longer communicated to surrounding cells, the production of hydroxyapatite decreases and osteoporosis (meaning "brittle bones") can result. Restoration of the piezoelectric signal can slow or reverse this condition.
Optimum waveforms, generated by alternating current signals, can nearly double the rate of bone healing in ordinary fractures, and restart healing in nonunion fractures, i.e. those fractures in which normal healing has stopped without rejoining the pieces of the broken bone. The conventional treatment of non-union fractures involves surgical procedures which are often unsuccessful and invariably increases both the discomfort and expense incurred by the patient. The use of electronic stimulation as a method of treating fractured bones has reduced the reliance on conventional surgery. See Donahue, R. C. T., et al, Optimization of electric field parameters for the control of bone remodeling: exploitation of an indigenous mechanism for the prevention of osteopenia. J Bone Miner Res 1993; 8 Suppl 2: S573-81; Chilibeck P D. Sale D G. Webber C E. Exercise and bone mineral density (1995) Sports Med; 19(2): 103-22.
Disorders of hard tissue
Except for the common cold, dental caries (tooth decay) is the most prevalent human disorder. See, The Merck Manual, Sixteenth Edition, p. 2480. Many steps have been taken to reduce dental caries and tooth decay, such as fluoridation and improved dental care, nevertheless tooth decay continues to be a significant problem. See, Featherstone, An Updated Understanding of the Mechanism of Dental Decay and its Prevention, Nutrition Quarterly, Vol. 14, No. 1, 1990, pp. 5-11.
To protect a normal tooth, a thin layer of dental enamel forms a protective coating over the tooth. This coating consists mainly of calcium, phosphate, and other ions in a hydroxyapatite-like structure. The enamel contains 2-5 percent carbonate; this carbonate content makes the enamel susceptible to acid dissolution. See, Featherstone, id. at 6.
Teeth vary considerably in their susceptibility to dissolution, and large crystallite size is correlated with resistance to dissolution (Besic et al, JADA, 91:594-601 (1975).
Bone formation and renewal
Bone is a highly specialized connective tissue with unique mechanical properties derived from its extensive matrix structure. A network of fibrous bundles composed of collagen is presumed to provide the tension-resistant behavior of bone. In addition, other materials including proteoglycans, noncollagenous proteins, lipids and acidic proteins associated with a mineral phase consisting primarily of poorly crystallized hydroxyapatite are deposited in the extensive matrix architecture of bone. Bone tissue is continuously renewed, by a process referred to as remodeling, throughout the life of mammals. This physiologic process might serve to maintain the properties of a young tissue.
The processes of bone formation and renewal are described by Mundy in "Bone Remodeling and Its Disorders" (1995, pub. Martin Dunitz). Osteogenesis vis-a-vis morphogenesis and growth of bone is presumably carried out by "osteoblasts" (bone-forming cells). Remodeling of bone is apparently brought about by an interplay between the activities of the bone-resorbing cells called "osteoclasts" and the bone-forming osteoblasts. The bony skeleton is thus not only an architectural structure with a mechanical function but also is a living tissue capable of growth, modeling, remodeling and repair. Since these processes are carried out by specialized living cells, chemical (pharmaceutical/hormonal), physical and physicochemical alterations can affect the quality, quantity and shaping of bone tissue.
The adult skeleton is composed of 80% cortical and 20% trabecular bone. Bone matrix is composed of 25% collagen, 65% inorganic material, and 10% non-cellular proteins (i.e., osteocalcin, silaoprotein, proteoglycans and osteonectin) and lipids. The term "mature bone" relates to bone that is mineralized, in contrast to non-mineralized bone such as osteoid. Osteoblasts synthesize and secrete Type 1 collagen and mucopolysaccharides to form the bone matrix which is laid down between the thin layers of osteoid. The layers are subsequently mineralized with 99% of the body's calcium found in the bone as a calcium phosphate complex with hydroxyapatite.
Conditions that affect bone strength
A number of dangerous and painful disabilities are caused by the excessive resorption of bone. For example, rheumatoid arthritis and periodontitis (both associated with erosive joint disease), osteoporosis, and the failure of prostheses to remain tightly bonded to the underlying bone are all characterized by the excessive resorption of existing bone. Medical research has studied such conditions for some time, but problems have not yet been resolved and treatment methods remain only partially satisfactory.
Osteoporosis is generally associated with a reduced trabecular bone volume leading to increased risk of bone fractures. This process is probably due to a metabolic imbalance between the rates of new bone formation and bone resorption. Osteoporosis can be divided into two classes: (1) type I or post-menopausal, which is related to reductions in estrogen content and affects primarily trabecular bone, and (2) type II or senile, which is related to reduced calcium absorption and affects primarily cortical bone.
Bone resorption can be divided into two processes which are probably being carried out concurrently. Phase I involves the inorganic metabolism conducted principally by osteoclasts, macrophages, monocytes, polymorphonucleocytes (PMNs), and fibroblasts. Osteoclasts are multi-nucleated cells which reabsorb calcium from bone and cartilage. This process is regulated by parathyroid hormone (PTH), Pg-E..sub.2, and cAMP which activate lysosomal hydrolytic enzymes and causes solubilization of the minerals in the bone, releasing calcium to the blood. Phase II involves organic metabolism where there is proteolytic destruction of the bone matrix collagen, releasing hydroxyproline to the blood. This process is initiated by the release of collagenase and cathepsin D from osteoblasts at the bone surface. The cellular enzymes belong to the metalloproteinase group of proteolytic enzymes which usually function at neutral pH. PTH binds to membrane receptors on osteoblasts, pre-osteoblasts and osteocytes, which activates the release of calcium from the dense bone, probably due to the activation of lysosomal enzymes, e.g., cathepsins, cAMP, interleukin-1, or prostaglandins.
Estrogen, progesterone, testosterone and vitamin D3 levels decrease with age. With advanced age there is a reduction of calcitonin that severely reduces calcium absorption from the gut. There is a positive correlation between the extracellular reduction of these physiological parameters and with osteoporosis. Other factors are: smoking, lack of exercise, sunlight, and disease states like myeloma, skeletal metastasis, gastric surgery, anti-convulsant therapy, male hypogonadism, thyrotoxicosis, amenorrhea, anorexia nervosa, hyperprolactinanemia, diabetes mellitus, immobilization, osteogenic imperfecta, and homocystinuria.
Existing treatments for apatite dissolution
Although a number of salts have been reported in certain experiments to counteract the dental decay process, no acceptable method of treatment using such salts, in the opinion of the inventor of the present invention, has been provided. One of the difficulties is providing a viable vehicle for delivering the salts. Still further, a number of safety issues are raised by some of the salts. Furthermore, sensory problems with respect to some of the salts prevent these salts from being taken on a regular basis by a patient to provide prophylactic benefits.
U.S. Pat. No. 5,378,131 provides a composition and method for preventing, or reducing the risk of, dental caries. A chewing gum is provided that includes a therapeutically effective amount of calcium glycerophosphate.
Calcium glycerophosphate counteracts the decay process. It is believed to function by reducing demineralization and/or increasing remineralization of tooth enamel. Chewing gum is an especially good delivery vehicle because it can deliver the ingredient over prolonged periods of time and can be conveniently used almost anywhere at anytime as opposed to a rinse or dentifrices. The method includes the step of adding to a sugar containing gum a sufficient amount of calcium glycerophosphate to offset the cariogenicity of the sugar present in the gum.
The apatite-forming-systems of U.S. Pat. No. 5,460,803, discussed previously, are described as useful in prevention and/or repair of dental weaknesses such as dental caries, exposed roots and dentin sensitivity.
Existing treatments for osteoporosis
A number of agents have been noted to attenuate loss of bone mass in elderly humans or to accelerate bone growth in the young, such as estrogens, insulin, fluorides, anabolic steroids, calcitonin, growth hormone, fibroblast growth factor, transforming growth factor, epidermoid growth factor, bone morphogenic protein (osteogenin), diphosphates, and oral calcium supplements, with varying degrees of success. See Rodan, G. A. (1995) Emerging therapies in osteoporosis, Annual Reports in Medicinal Chemistry, 29: 275-285. Most evidence indicates that massive intakes of calcium (1500-2000 mg/day) orally does not prevent bone loss in post-menopausal women. It is not clear what the mechanism of action of estrogen is in blocking bone resorption.
It is clear, however, that the activity of osteoblasts and osteoclasts is coordinated and regulated by a complex mechanism and is affected by a variety of hormones and prostaglandins. See Raisz et al., Ann. Rev. Physiol., 43:225 (1981); U.S. Pat. No. 4,921,697 which teaches that inhibition of prostaglandin production by IFN-gamma is a treatment for osteoporosis and other bone-resorption diseases.
It is known that very little control is possible over the duration and the concentration at which prostaglandins reach the bone cells. It is also known that systemic injection or infusion of prostaglandins is an alternative with significant drawbacks since the lungs efficiently remove prostaglandins from circulation. See W. Harvey and A. Bennett, "Prostaglandins in Bone Resorption" CRC Press, pp. 37 (1988).
Frost et al. in "Treatment of Osteoporosis by Manipulation of Coherent Bone Cell Populations," Clinical Orthopedics and Related Research, 143, 227 (1979) discloses a theoretical model that suggests it should be possible to synchronize the activity and metabolism of bone cells by administering bone cell activating agents first and then administering a bone resorption inhibiting agent. This proposed model assumes that bone formation inhibition does not take place, because no bone resorption inhibiting agent is administered during the bone formation phase of the bone remodeling unit. EPO App. No. 0 381 296 teaches the use of a kit wherein a bone activating period or treatment regime is followed by a bone resorption inhibiting regime. Many examples of bone activating compounds are cited in this reference. See also U.S. Pat. No. 5,118,667.
Uses of synthetic apatite
Apatite preparations have been proposed for use as bone inductors (to induce bone formation) and osteoconductors (by acting as scaffolds to facilitate continuous progression of new bone formation). These apatite preparations are mostly of synthetic origin and distinct structurally and chemically from the biological calcium-phosphate crystals in bone. All of these apatites are not only chemically and structurally distinct from the apatite crystals of bone, especially in their short range order, size and reactivity, but in some cases, they contain varying amounts of amorphous calcium-phosphate, that is, calcium-phosphate solids which are not crystalline at all. In other instances, the calcium-phosphates made synthetically also contain calcium salts other than apatite crystals such as calcium oxides.
Other uses of apatite are diverse. For example, U.S. Pat. No. 05,427,754 to Nagata et al. issued Jun. 27, 1995 describes the use of apatite as an adsorbent in chromatography. The kind of protein adsorbed on hydroxyapatite varies with the kind of crystal faces of the hydroxyapatite. It is, therefore, necessary to produce hydroxyapatite which possesses crystal faces befitting the particular kind of protein to be adsorbed.
U.S. Pat. No. 05,405,436 to Raab et al. issued Apr. 11, 1995 describes a process for preparation of a hydroxyapatite suitable as an abrasive tooth-cleaning substance.
Uses of natural apatite
Living organisms are also apatite-forming-systems. In recent years, interest has been shown in the osteoconductive properties of a physiologically formed hydroxyapatite substratum that is obtained from living organisms. Thus, U.S. Pat. No. 5,439,951 to Glimcher et al. issued Aug. 8, 1995 provides a process for isolating from the biologically, naturally formed crystals of bone a purified apatite that is substantially free of organic material. Another example is a porous hydroxyapatite substratum that is obtained after hydrothermal conversion of the calcium carbonate exoskeletal microstructure of the scleractinian reef-building corals, Porites and Goniopora. This hydroxyapatite is characterized by a relatively uniform network of interconnected channels and pores, similar to the mineralized inorganic supporting structure of living bone. Experimental evidence has established the osteoconductive properties of the porous substratum when it is implanted in orthotopic sites, and the material has been used experimentally in reconstructive operations, particularly craniofacial procedures, as an alternative to autogenous bone grafts. Bone forms in porous hydroxyapatite that has been implanted extraskeletally in non-human primates. The shape and configuration (hereinafter referred to as "the geometry") of the porous hydroxyapatite substratum can be a relevant factor in determining the osteoconductive potential of hydroxyapatite. U.S. Pat. No. 05,355,898 provides evidence that the geometry of a substratum can be critical for inducing bone growth.
No general conclusions can be drawn from these representative reports except that the need for materials which are useful in fixation of implants and in repair or replacement of hard tissue defects remains and that the materials now available do not solve the many problems associated with the treatment of these problems, due to many variables, including the properties of the materials as well as the ease with which they can be manufactured and utilized.
Other hard tissue related problems
Bone mass is decreased by treatment with the following drugs over a long period of time: glucocorticoids, thyroxine, heparin, cytotoxic drugs, retinoids vit A!, phorbol esters, Pg-E's, interleukin-1, endotoxins and PTH.
A number of dangerous and painful disabilities are caused by the excessive resorption of bone. For example, rheumatoid arthritis and periodontitis (both associated with erosive joint disease), osteoporosis, and the failure of prostheses to remain tightly bonded to the underlying bone are all characterized by the excessive resorption of existing bone. For a discussion of treatment methods, see Reports in Medicinal Chemistry 29: 275-285. Medical research has studied such conditions for some time, but problems have not yet been resolved and treatment methods remain only partially satisfactory.
OBJECTS AND ADVANTAGES
The present invention is directed to processes and products that satisfy the need to (1) mitigate the effects of hard tissues diseases; (2) provide apatite compositions suitable for a variety of uses. Embodiments of the present invention significantly mitigate the effects of many hard-tissue diseases by providing methods and compositions for strengthening apatite in teeth and in bones. One manifestation of the improved apatite is larger crystallite size. Larger crystallite size confers resistance to dissolution. An understanding of the inverse relationship between crystallite size and dissolution rate helps to predict the properties of apatite having a large crystallite size. The present invention is constituted, however, without limitation by a relationship between crystallite size and the dissolution rate of apatite.
An embodiment of the present invention significantly mitigates the effects of excessive bone resorption and spares the use of other drugs presently used for mitigation, by providing a method to form stronger bones.
Many forms and uses of apatite exist, and examples of both have been cited herein. The differences between the various forms may be subtle; nevertheless, each difference alters the suitability of the apatite for a given purpose. Use of apatite in chromatography for example, requires apatite which possesses crystal faces befitting the particular kind of protein to be adsorbed. The present invention provides methods for producing forms of apatite. The new forms of apatite provided by the present invention could be useful in the manufacture of abrasives such as tooth pastes, osteoconductors, bone inductors, chromatographic media, and remineralizing agents, particularly for dental lesions. The possible forms of apatite provided by the present invention is not limited to the examples, but may include other forms as the need arises.